PROTEIN PHOSPHATASE ASSAY USING A MODIFICATION OF THE P81 PAPER PROTEIN-KINASE ASSAY PROCEDURE

Synthetic peptides have been used to define specificity determinants a nd to distinguish reactivities of numerous protein kinases and phospho protein phosphatases. Direct analysis of peptide phosphorylation is mo st often determined using P81 phosphocellulose paper to separate modif ied peptide and unreacted [gamma-P-32]ATP; however phosphopeptide deph osphorylation is usually determined by extraction and quantitation of phosphomolybdate complexes or ion exchange chromatography. We describe here the adaptation of the rapid, direct P81 paper protein kinase ass ay for the determination of phosphopeptide dephosphorylation. The S6-2 1 peptide (AKRRRLSSLRASTSKSESSQK), which is derived from the multiphos phorylated carboxyl terminal domain of the S6 ribosomal protein, was p hosphorylated by a human placenta S6 kinase and dephosphorylation by p urified phosphoprotein phosphatase type 1 in the presence of a variety of buffers, and inhibitors/activators was determined using the new as say. Results comparable to those obtained with the ion-exchange chroma tography were obtained. and the assay was significantly less expensive , more rapid. and more accurate than methods previously used to quanti tate phosphopeptide dephosphorylation.